This invention relates to hepatitis B virus (xe2x80x9cHBVxe2x80x9d) core antigen particles that are characterized by multiple immunogen specificities. More particularly, the invention relates to HBV core antigen particles comprising immunogens, epitopes, or other related structures, crosslinked thereto by ligands which are HBV capsid-binding peptides that selectively bind to HBV core protein. Such particles may be used as delivery systems for a diverse range of immunogenic epitopes, including the HBV capsid-binding peptides, which advantageously also inhibit and interfere with HBV viral assembly by blocking the interaction between HBV core protein and HBV surface proteins. Mixtures of different immunogens, HBV capsid-binding peptide ligands, or both, may be crosslinked to the same HBV core particle. Such resulting multicomponent or multivalent HBV core particles may be advantageously used in therapeutic and prophylactic vaccines and compositions, as well as in diagnostic compositions and methods using them.
The front-line of clinical immunotherapeutic regimens includes patient immunizations against infectious pathogens and other health-threatening agents. Despite the plethora of immunization agents, inoculations may afford, at best, partial immunity, requiring frequent re-immunizations. Such is the case for various conventional monovalent or polyvalent vaccines. And even among such vaccines, the number of single agent inoculants capable of eliciting immunity against a variety of immunogens is limited. Furthermore, antigenic variation among pathogens may limit the efficacy of conventional vaccines.
Due to such obstacles, efforts have focused on methodologies for enhancing the immune system response to given immunogens. To that end, immunogenic conjugates have been produced by linking immunogens to hepatitis B virus (xe2x80x9cHBVxe2x80x9d) core particles (also referred to as nucleocapsids or nucleocapsid shells), in efforts to enhance the immunogenicity of the linked immunogen, through the operation of T cell dependent and T cell independent determinants of HBV core antigen. See, for example, U.S. Pat. No. 4,818,527 and R. Ulrich et al., xe2x80x9cCore Particles of Hepatitis B Virus as Carrier for Foreign Epitopesxe2x80x9d, Adv. Virus. Res., 50, pp. 141-82 (1998). Enhanced immunogenicity of epitopes of interest has also been approached via hybrid viral particle-forming proteins, comprising at least a portion of a naturally occurring viral particle forming protein, for example HBV surface antigen, and one or more epitopic sites of interest. See U.S. Pat No. 5,965,140. As evident from such efforts, proteins of HBV have been used as platforms for presenting immunogens of interest to the immune system.
Hepatitis B virus is a blood-borne virus, comprising a small, partially double-stranded DNA genome, carrying four extensively overlapping open reading frames, consisting of an inner nucleocapsid, comprising the HBV core protein (xe2x80x9cHBcAgxe2x80x9d), viral polymerase and viral DNA, surrounded by a membranous envelope containing HBV surface antigens (xe2x80x9cHBsAgxe2x80x9d). The viral envelope contains three different, but related surface antigen proteins, long (L), medium (M) and short (S), which share a common carboxy terminal region but have different amino termini, arising from variable use of initiation triplets at different points within a continuous open reading frame.
The long polypeptide (L polypeptide) consists of pre-S1, pre-S2 and S regions. It is the product of the entire reading frame and comprises the pre-S1 domain of 108 amino acids (or 199, depending on the virus subtype) at its amino terminus, followed by the pre-S2 domain of 55 amino acids, and the short polypeptide (S polypeptide) region of 226 amino acids. The medium-length polypeptide (M polypeptide) has the pre-S2 domain at its amino terminus followed by the S region, whereas the S polypeptide, which is the most abundant form, consists of only the S region. The pre-S regions are believed to play an important role in both viral assembly and attachment to the host cell. The S form is more abundant than the M and L forms of HBsAg in the virus, and occurs in both glycosylated and nonglycosylated forms [V. Bruss and D. Ganem, xe2x80x9cThe Role of Envelope Protein in Hepatitis B Virus Assemblyxe2x80x9d, Proc. Natl. Acad. Sci. USA, 88, pp. 1059-63 (1991); V. Bruss et al., xe2x80x9cPost-translational Alteration in Transmembrane Topology of Hepatitis B Virus Large Envelope Proteinxe2x80x9d, EMBO J., 13, pp. 2273-79 (1994); A. R. Neurath et al., xe2x80x9cIdentification and Chemical Synthesis of a Host Cell Receptor Binding Site on Hepatitis B Virusxe2x80x9d, Cell, 46, pp. 429-36 (1986); K. Ueda et al., xe2x80x9cThree Envelope Proteins of Hepatitis B Virus: Large S, Middle S and Major S Proteins Needed for the Formation of Dane Particlesxe2x80x9d, J. Virol., 65, pp. 3521-29 (1991)]. Specific interactions between the outer surface of the core and the inner surface of the envelope are likely to guide correct assembly of the virus and stabilize the resulting particle
HBV core protein can be expressed efficiently in E. coli [M. Pasek et al., xe2x80x9cHepatitis B Virus Genes and Their Expression in E. colixe2x80x9d, Nature, 282, pp. 575-79 (1979)], where it assembles into icosahedral shells of two sizes containing either 180 (T=3) or 240 (T=4) subunits [R. A. Crowther et al., xe2x80x9cThree-Dimensional Structure of Hepatitis B Virus Core Particles Determined by Electron Microscopyxe2x80x9d, Cell, 77, pp. 943-50 (1994)]. The subunits are clustered as dimers and each dimer forms a spike which protrudes on the surface of the shell. Using electron cryomicroscopy and image processing, a map of the T=4 shell was recently made at 7.4 xc3x85 resolution from images of more than 6000 individual particles [B. Bxc3x6ttcher et al., xe2x80x9cDetermination of the Fold of the Core Protein of Hepatitis B Virus by Electron Cryomicroscopyxe2x80x9d, Nature, 386, pp. 88-91 (1997)]. This revealed the fold of the polypeptide chain, which was largely xcex1-helical and quite unlike previously solved viral capsids. Each dimer spike was formed by a pair of long xcex1-helical hairpins, one from each monomer in the dimer [Bxc3x6ttcher et al. (1997); J. F. Conway et al., xe2x80x9cVisualization of a 4-Helix Bundle in the Hepatitis B Virus Capsid by Cryo-electron Microscopyxe2x80x9d, Nature, 386, pp. 91-94 (1997)]. A numbering scheme which superimposed the amino acid sequence on the fold [Bxc3x6ttcher et al. (1997)] placed the major immunodominant region of the HBV core protein around amino acids 78-82 [J. Salfeld et al., xe2x80x9cAntigenic Determinants and Functional Domains in Core Antigen and E Antigen from Hepatitis B Virusxe2x80x9d, J. Virol, 63, pp. 798-808 (1989); M. Sxc3xa4llberg et al., xe2x80x9cCharacterisation of a Linear Binding Site for a Monoclonal Antibody to Hepatitis B Core Antigenxe2x80x9d, J. Med. Virol., 33, pp. 248-10 52 (1991)], at the tip of the spike.
Agents which inhibit HBV viral assembly include those that bind to the core antigen of HBV, thereby blocking the interaction between HBV core proteins and HBV surface proteins. Some such HBV capsid-binding peptides are described in PCT patent application WO98/18818 and in M. R. Dyson and K. Murray, xe2x80x9cSelection of Peptide Inhibitors of Interactions Involved in Complex Protein Assemblies: Association of the Core and Surface Antigens of Hepatitis B Virusxe2x80x9d, Proc. Natl. Acad. Sci. USA, 92, pp. 2194-98 (1995).
As will be apparent from the disclosure to follow, HBV capsid-binding peptides may be advantageously used as ligands for constructing HBV core antigen particles characterized by the ability to elicit enhanced immune responses to single or multiple immunogens.
The present invention addresses the problems referred to above by providing HBV core antigen particles which elicit enhanced immunogenicity to one or more component immunogens. Such multicomponent or multivalent HBV core antigen particles comprise immunogens, epitopes, or other related structures, crosslinked thereto through ligands which are peptides that selectively bind to HBV core antigen particles, in addition to immunogenic domains or epitopes attached to or inserted into the HBV core antigen polypeptide via genetic manipulation of the coding sequence or by polypeptide synthesis. Such particles may be used as delivery systems for a diverse range of immunogenic epitopes, including the HBV capsid-binding peptides, which themselves, inhibit and interfere with HBV viral assembly by blocking the interaction between HBV core protein and HBV surface proteins. The resulting multicomponent or multivalent HBV core particles may be advantageously used in therapeutic and prophylactic vaccines and compositions, as well as diagnostic compositions and methods using them.
The present invention advantageously permits mixtures of different immunogens, HBV capsid-binding peptide ligands, or both, to be crosslinked to the same HBV core particle. The result is single particles that are efficient stimulants of T cells and which are immunologically multivalent. Thus, a single antigen-presenting cell can stimulate the proliferation of multiple B cell clones of differing specificity.